Sequencing of the human genome, and the genomes of other species, has emphasized the fact that the expression and properties of a protein are often dependent on posttranslational modifications and, thus, cannot be predicted from the DNA sequence. This realization has spurred an interest in proteomics, the study of protein expression within a cell under defined conditions.
Traditionally, proteins from biological samples have been isolated and identified by separating the proteins using 2-D gel electrophoresis followed by identification of the protein using mass spectrometry. However, this method is time consuming and can only detect proteins that are highly abundant in the biological sample. Severe streaking causes deterioration in resolution of the electrophoretic separation when high loading is used in an attempt to visualize less abundant proteins.
Particular difficulties have been encountered in attempts to use 2-D gel electrophoresis/mass spectrometry to study glycosylated proteins, as they are often present in low abundance. Glycosylated proteins are of particular interest in proteomic studies, however, as such proteins often participate in signal transduction and other cellular processes.
Glycosylated proteins and peptides (also referred to herein as glycoproteins and glycopeptides, respectively) are normally isolated from mixtures using different lectin affinity columns. For example, a BS-II (Bandeiraia simplicifolia) column has a high specificity for peptides having O-linked N-acetyl glucosamine (O-GlcNAc) moieties. See Apffel et al. (1996) J. Chromatogr. A 732: 27-42. Geng et al. have reported using lectin affinity column chromatography to recover glycopeptides, followed by isotope tagging of the glycopeptides with generic N-terminal labels. See Geng et al. (2000) J. Chromatogr. A 870: 295-313.
Alternatively, the selective and sequential use of enzymes can cleave sugars, and the before and after results can be analysed, for example, by tandem mass spectrometry. See Geng et al. (2001) J. Chromatogr. B 752: 293-306. O-linked glycopeptides can be deglycosylated using O-glycosidase.
The mapping of O-GlcNAc sites after beta-elimination has been reported by Wells et al. See Wells et al. (2002) Molec. & Cell. Proteomics, 1.10, 791-804. After the beta-elimination reaction, Wells et al. used solution phase Michael-addition chemistry to label glycosylation sites of proteins with either dithiothreitol or biotin pentylamine and analyzed the products using mass spectrometry. The methods described by Wells et al. involved multiple purification and clean-up steps to provide the ultimate product.
While the above methods have greatly facilitated the study of glycoproteins, they have various disadvantages, such as requiring multiple purification or clean-up steps in isolating or labeling a target protein or collection of proteins. Thus, a method that does not suffer from the disadvantages of the above methods is desirable.